1. Field of the Invention
The present invention relates generally to the fields of drug action, cellular regulation and gene therapy. More specifically, the present invention relates to the novel finding that glucocorticoids enhance reporter gene activity when transfected by cationic lipid or liposomes.
2. Description of the Related Art
Human clinical trials for gene therapy as treatment of diseases caused by genetic defects or by gene dysregulation have shown promise and gained momentum in the last two years. Several diseases which manifest significant symptomology in the lung have been targeted for gene therapy, including cystic fibrosis and lung cancer. These trials have employed either recombinant retroviral or, adenoviral vectors as well as cationic lipids to transport and deliver the gene to the cell. Nonetheless, cellular transfer and subsequent gene expression is low and thus therapeutic levels of gene expression may be lacking. Moreover, immune responses developing against viral vectors may limit their use. While cationic lipids are less efficient at delivery than the adenoviral vectors, newer chemical designs have produced cationic lipids that are greatly improved over the original designs. Several animal and human trials have shown that, at cationic lipid concentrations typical for transfection, no negative side effects or immune response developed.
Delivery of gene therapy to the lung by aerosol allows genes to be delivered directly to the target tissue. Several groups have demonstrated aerosol delivery and transfection of animal lungs in vivo using reporter gene DNA coupled to cationic liposomes. The salient features noted in these studies were the absence of toxicity and a duration of gene expression of approximately 1 month. Gene expression was still relatively low; even modest transfection in mouse lungs required at least 0.5 to 12 milligrams of highly purified DNA. As reported, gene therapy by these methods would not be feasible for humans.
Another approach to increasing transfection efficiency is a greater understanding of plasmid uptake and the factors which influence expression of the transfected gene in the target tissue. In recent investigations as to the role of inflammation on gene transfection in lung cells, it was found that an exposure of A549 human lung carcinoma cells to the immune stimulator lipopolysacchride or the cytokine IL-1xcex2 before transfection with pCMVxcex2gal-DMRIE/DOPE reduced the level of xcex2-gal protein below that seen in the cells treated with medium only.
The prior art is deficient in the lack of effective means of delivering therapeutic levels of transfected genes. The present invention fulfills this longstanding need and desire in the art.
Little is known about: the mechanism by which cationic lipid-DNA complexes are taken up by the cell or, the fate of the complexes within the cell. Even less is known about factors in situ that may influence uptake, or the consistent delivery and expression of DNA once in the tissue, especially in patients with chronic lung inflammatory disease or any other immune process. The present invention demonstrates two findings which have a substantial effect on tranfection in cell culture and which have parallels in vivo. First, the present invention demonstrates that the cytokine IL-1xcex2 and the immune stimulator lipopolysacchride (LPS) suppress transfection/expression of pCMVxcex2gal transfected into A549 human lung cancer cell lines or primary rat lung cells by cationic lipid. Secondly, anti-inflammatory topical glucocorticoids such as beclomethasone dipropionate (BEC) reverse the inhibitory effects of IL-1xcex2 and lipopolysacchride and even enhance expression of reporter genes above and beyond expression seen in untreated transfected cells, i.e., not treated with lipopolysacchride or IL1xcex2, etc. The effect is specific to glucocorticoids, as opposed to other types of steroids but not to a particular glucocorticoid. This effect may also be specific to glucocorticoids as anti-inflammatory agents as this effect was not seen when cells were pre-treated with another immunosuppressant cyclosporine A. Glucocorticoid-mediated boost in transgene activity is independent of promoter, reporter gene and cationic lipid used. The mechanism by which glucocorticoids enhanced expression of reporter genes does not involve increased plasmid-lipid uptake, but rather an intracellular mechanism which does not involve new protein synthesis. In addition, pretreatment of primary rat lung cells with synthetic topical glucocorticoids, in the absence of lipopolysacchride or IL-1xcex2, prior to transfection enhanced the level of xcex2-gal protein over untreated controls. The present invention describes studies concerning the mechanism of glucocorticoid-enhanced transfection of plasmid DNA. Thus, the present invention has direct relevance to the use of gene therapy in vivo.
In one embodiment of the present invention, there is provided a method of increasing the cellular expression of a gene in a biological tissue after delivery of said gene in an appropriate vector to an animal, comprising the step of: administering to said animal a pharmacologically effective dose of a glucocorticoid in an amount sufficient to increase the cellular expression of said gene.
In another embodiment of the present invention, there is provided a method of treating a pathophysiological state in a human by increasing the cellular expression of a gene in an appropriate vector after delivery of said gene into a biological tissue of a human in need of such treatment, comprising the step of: administering to said human a pharmacologically effective dose of a glucocorticoid in an amount sufficient to increase the cellular expression of said gene.